agarose gel electrophoresis - assiut university electorphoresis seleem.pdfagarose gel...

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Agarose Gel Electrophoresis Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS- PAGE used for protein analyses) Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. We will be using agarose gel electrophoresis to determine the presence and size of PCR products.

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Page 1: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Agarose Gel Electrophoresis

• Gel electrophoresis is a widely used technique for the analysis of

nucleic acids and proteins. Agarose gel electrophoresis is

routinely used for the preparation and analysis of DNA. (SDS-

PAGE used for protein analyses)

• Gel electrophoresis is a procedure that separates molecules on

the basis of their rate of movement through a gel under the

influence of an electrical field.

• We will be using agarose gel electrophoresis to determine the

presence and size of PCR products.

Page 2: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

http://gslc.genetics.utah.edu/units/biotech/gel/

Virtual Gel Electrophoresis

Additional Information on

Gel Electrophoresis:

Page 3: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

• DNA is negatively charged.

+-

Power

DNA

• When placed in an electrical field, DNA will migrate toward the positive

pole (anode).

H

O2

• An agarose gel is used to slow the movement of DNA and separate by size.

Scanning Electron Micrograph

of Agarose Gel (1×1 µm)

• Polymerized agarose is porous,

allowing for the movement of DNA

Page 4: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

+-

Power

DNA

How fast will the DNA migrate?

strength of the electrical field, buffer, density of agarose gel…

Size of the DNA!

*Small DNA move faster than large DNA

…gel electrophoresis separates DNA according to size

small

large

Page 5: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for
Page 6: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Agarose

Agarose is a linear polymer extracted from seaweed.

D-galactose 3,6-anhydro

L-galactose

•Sweetened agarose gels have

been eaten in the Far East since

the 17th century.

•Agarose was first used in biology

when Robert Koch* used it as a

culture medium for Tuberculosis

bacteria in 1882

*Lina Hesse, technician and illustrator for

a colleague of Koch was the first to

suggest agar for use in culturing bacteria

Page 7: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Making an Agarose Gel

Page 8: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

An agarose gel is prepared

by combining agarose

powder and a buffer

solution.

Agarose

Buffer

Flask for boiling

Page 9: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Casting tray

Gel combs

Power supply

Gel tank Cover

Electrical leads

Electrophoresis Equipment

Page 10: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Gel casting tray & combs

Page 11: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Seal the edges of the casting tray and put in the combs. Place the casting

tray on a level surface. None of the gel combs should be touching the

surface of the casting tray.

Preparing the Casting Tray

Page 12: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Agarose Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is

several times larger than the volume of buffer.

Page 13: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Agarose is insoluble at room temperature (left).

The agarose solution is boiled until clear (right).

Gently swirl the solution periodically when heating to allow all the grains of agarose

to dissolve.

***Be careful when boiling - the agarose solution may become superheated and

may boil violently if it has been heated too long in a microwave oven.

Melting the Agarose

Page 14: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Allow the agarose solution to cool slightly (~60ºC) and then carefully

pour the melted agarose solution into the casting tray. Avoid air

bubbles.

Pouring the gel

Page 15: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Each of the gel combs should be submerged in the melted agarose solution.

Page 16: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

When cooled, the agarose polymerizes, forming a flexible gel. It should

appear lighter in color when completely cooled (30-45 minutes).

Carefully remove the combs and tape.

Page 17: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Place the gel in the electrophoresis chamber.

Page 18: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

buffer

Add enough electrophoresis buffer to cover the gel to a depth of

at least 1 mm. Make sure each well is filled with buffer.

Cathode

(negative)

Anode

(positive)

wells

DNA

Page 19: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

6X Loading Buffer:

Bromophenol Blue (for color)

Glycerol (for weight)

Sample Preparation

Mix the samples of DNA with the 6X sample loading buffer (w/ tracking

dye). This allows the samples to be seen when loading onto the gel, and

increases the density of the samples, causing them to sink into the gel

wells.

Page 20: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample.

The sample should sink into the well. Be careful not to puncture the

gel with the pipette tip.

Page 21: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Place the cover on the electrophoresis chamber, connecting the electrical

leads. Connect the electrical leads to the power supply. Be sure the leads

are attached correctly - DNA migrates toward the anode (red). When the

power is turned on, bubbles should form on the electrodes in the

electrophoresis chamber.

Running the Gel

Page 22: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

wells Bromophenol Blue

Cathode

(-)

Anode

(+)

Gel

After the current is applied, make sure the Gel is running in the correct

direction. Bromophenol blue will run in the same direction as the DNA.

DNA

(-)

Page 23: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

100

200 300

1,650

1,000

500

850

650

400

12,000 bp

5,000

2,000

DNA Ladder Standard

Inclusion of a DNA ladder (DNAs of know sizes) on the gel

makes it easy to determine the sizes of unknown DNAs.

-

+

DNA

migration

bromophenol blue

Note: bromophenol

blue migrates at

approximately the

same rate as a 300

bp DNA molecule

Page 24: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

As an alternative to purchasing costly DNA ladders, one can be created

using meal worm (Tenebrio molitor) DNA and a restriction enzyme.

http://people.uis.edu/rmosh1/DNAexerciseVIIa02.pdf

Page 25: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Staining the Gel

***CAUTION! Ethidium bromide is a powerful mutagen and is

moderately toxic. Gloves should be worn at all times.

• Ethidium bromide binds to DNA and fluoresces under UV light,

allowing the visualization of DNA on a Gel.

• Ethidium bromide can be added to the gel and/or running buffer

before the gel is run or the gel can be stained after it has run.

Page 26: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Safer alternatives to Ethidium Bromide

Methylene Blue

BioRAD - Bio-Safe DNA Stain

Ward’s - QUIKView DNA Stain

Carolina BLU Stain

…others

advantagesInexpensive

Less toxic

No UV light required

No hazardous waste disposal

disadvantagesLess sensitive

More DNA needed on gel

Longer staining/destaining time

Page 27: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Staining the Gel

• Place the gel in the staining tray containing warm diluted stain.

• Allow the gel to stain for 25-30 minutes.

• To remove excess stain, allow the gel to destain in water.

• Replace water several times for efficient destain.

Page 28: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Ethidium Bromide requires an ultraviolet light source to visualize

Page 29: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Visualizing the DNA (ethidium bromide)

100 200 300

1,650 1,000

500

850 650

400

5,000 bp 2,000

DNA ladder

DNA ladder

PCR Product

1 2 3 4 5 6 7 8

wells

+ - - + - + + -

Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA

Primer dimers

Page 30: Agarose Gel Electrophoresis - Assiut University Electorphoresis seleem.pdfAgarose gel electrophoresis is routinely used for the preparation and analysis of DNA. (SDS-PAGE used for

Visualizing the DNA (QuikVIEW stain)

250

1,500 1,000

500 750

2,000 bp

DNA ladder

PCR

Product

wells

+ - - - - + + - - + - +

March 12, 2006

Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA